Process for purifying enzymes



smaller molecules, both organic, and impurities are often removed byalcohol, ammoniurn'jsul 3,208,918 PROCESS FOR PURIFYING ENZYME RolandFrank Beers, Jr., 1406 Carrollton Ave., Baltimore, Md. No Drawing. FiledAug. 15, 1963, Ser. No. 302,457

I Claims. (Cl. 195-66) This invention relates to an enzyme purificationprocess. More particularly, this invention concerns a process for thepurification of enzymes useful in the synthesis of polynucleotides.

In accordance with this invention, a method is provided whereby enzymesuseful in the synthesis of polynucleotides may be prepared substantiallycompletely free from any detectable quantities of contaminating enzymes.In particular, the invention provides a method for the separation ofenzymes from complex protein mixtures containing, for example,polynucleot'ide phosphorylase, phosphodiesterases, phosphatases,adenylate kinase and the tion techniques, the processes have been workedout in. a

more or less trial and error fashion according to the specific enzymedesired. Some of the more common purification methods'used are, (1)maceration of tissue with an abrasive material, e.g.,.sand. or highspeed, specially designed pulverizers; (2) alternate freezing andthawing techniques;

(3) dehydration, followed by grinding, leaving the material in an easily.extractable form; and (4) autolysis. Water, glycerine,-.and variousbuffer solutions have been f United States Patent 0 ofpolyribonucleotides, for example, polyadenylic' employedto dissolve theenzymes liberated by methods such as the foregoing, followed byinorganic. Pr'otein fate, or isoelectric precipitation. Attempts havealso been made to precipitate .the enzyme leaving the bulk of theimpurities in solution. None of the foregoing methods have successfullypurified enzymes completely free of contaminants.

Adsorption and elution procedureshave also playedan important role inenzyme purification, Under controlled conditions of pH, temperature andconcentration, various adsorbents, for example, alumina and kaolin,preferentially adsorb certain molecules. By changing one, or

. more of the conditions, usually the pH, varying degrees of successhave been achievedby eluting the adsorbed molecules into pure solventAll of these" known, techniques are; relatively non-specific in/theirvaction and are usually laborious and require complicated and expensivelaboratory equipment. Infmany. instances these'techniques have beenincompletely successful in the purifica-- tion of the enzymes. In theusual instance, the activity dialysis to eliminate adenosinediphosphate. so

' I material, polyadenylic acid, l approximately 0.4 milliequivalent ofpolyadenyli'c acid of the' enzyme is greatly lessenedland-rarelyis theenzyme produced free'of contaminating enzymes.

Broadly, the method of thisinvention comprises forming a complex betweenthe desired enzyme in the mixture to be purified, and a; specificsubstrate for the enzyme. The inventive concept for this inventiontakesadvantage of the high degree of specificity of the enzyme for itssubstrate which results inithe formation of an enzymesubstrate complex.It is theorized that the spatial conpolymer having a concentration of 2figuration and arrangement cules are the important criteria indeterminin'g the specificity of many and perhaps all of the enzymes.

The term substrate, as referred to in-this application,

includes the specificsubstance acted on by, a'lspecific enzyme. By wayofexample of specific substratemate rial suitable for use in the methodof this inventionv are the series of substances belonging to the generalclass acid I 'polyuridylicacid, .polyinosinic acid and polycytidylicacid; Numerous of these polyr-ibonucleotide substrates have beenprepared synthetically and have found quite suitable in the-method .ofthis invention.

A combining" a' substrate material' as defined above, with an insolublematerial, contactingjthe enzyme mixture to be purified with thesubstrateinsoluble material'complex to form a specific enzyme'esubstratecomplex and thereafter eluting the enzyme from-1 the substrate materialand subsequently separating the'highly purified enzyme from the eluatesolution. invention thatthe affinityofthe, substra'tematerial and theinsoluble material be-gr'eat'enough to prevent elution byv theelutrient. A suitable insoluble'material useful as adsorbent. is thecellulose resin,'diethylaminoethylcel- Iulose (DEAE-cellulose)'.Carboxymethyl cellulose,

'phosphoric acid-cellulose, and'a composition made from epichlorohydrin,triethanolamine and -cellulose (ECTEOLA-cellulose) bind poly A. very1oosely and are not as effective.

ample Dowex 'l-x'8, Dowex' 2 x8 and Amberlite .IR400 also bindpolynucleotides, but. during elutio'n of .,the-

enzymes thesepolymers also are eluted. -As pointed out above, it isessential. that theabsorbent or-insolublematerial be .ofa character thathas a high degree of alfinity for the substrate material.

- The following exampleswill illustrate the purification of specificenzymes utilizingthe method of this invention; however, it is'to'beunderstood'that the examples are not to be consideredaslimitations uponthe scope of the inventive concepts.

" .PREPARATTONSOHF SUBSTRATE-ADSORBENT COLUMN h Erearripl I purifiedpreparations of Polyadeny lie acid synthesized by the action-ofpartially polynucl'eotide phosphorylase on was used. as. a substrate.The polymer"-was freed' of contaminating proteins by. known techniques.The. purified polymer was dialyzed against distilled water'for. 24 hoursat 4 C. A solution of the about00l M. organic phosphate 'was applied toa column of 'diethylaminoethylcellulose used as -the adsorbent material.

The adsorbent containing 0.-8'l milliequivalent per gram when suspended-'in water bound per gram of cellulose, or

per gram ofcellulose. I

The so'lution'of substrate materialwas allowed to flow through theadsorbent until the concentration of the substrate in the eluate wasequal to the concentration of the substrateappliedto the column. Thecolumn was then washed with water until the eluate was free of substratematerial.

The column of substrate-adsorbent material was then washed with a 1.0 M.solution of potassium chloride, pH

8, buffered with 0.01 M. tris (hydroxymethyl) aminomethane (tris buffer)to remove 'any short chain polymers poorly adsorbed to the column. Itwill beappreciated that any monovalent salt is suitable for use inremoving Patented Sept. 2 8, 19 65 of individual substrate moleit beenspecific .embodiment of the; inventioncomprises 'It is importantpin themethod of this' Certain exchange resins, for ex- 0.12g. of the substratei hai-sani y; ashesquanfi yggheiizym pa tas v 1 en'z' meaaeasa I Ithorough] 4 1,500 m for ADI-minutes; I

"rate of lysis of the cells by move any of the polyadenylic :acidfromthe column.

short chain: polymers. Repeated washing failedto re- Repeatedfr'actionations' of variousfenzyme preparations also failed toremovexa'n'ylof the substrate material. Any of the-well knownchromatographic column techniques may be used inathe method of -thisiinv entin;' Care Prior; to 'each fractionation,-; thesubsti'titeadsorbcnt no ultra-violet adsorbentfrnateriai (260 mu,"

aminoethylcellulo'se'is used; 'as", the' adsorbent). I 'EN YME'A SS Y vprocess of this invention was 'determinedg-as follows g mixtnrelis 'preparedby adding v to' aclinicai centrifuge tube 0.5 ml. of .e'ch ofthefollowing: 2 0.01' M: magn siumfihl r (3) .0.1M.-tris',.pH 9.5 v j.(4) 1. M.- potassiumchloride j of; the opium alternative method is todialyze the solution against the buffer. untilthe-ammonium sulfate isremoved. I:

'A preliminary fractionation of the out on diethy-laminoethylcellulose,v with TEM- butter,

enzyme was carried previously "Washed The enzyme preparation was addedto a column untilapproximately:50% .oiE'thev upper half was st'ainedbrownby the, contaminating catalas'e 'A linear gradient ofKCYin TEMbufler was lien zfipplied. The 'steepness "of'the-g'radient is, ofcourse, dete'rrnifn'ed by-the dimensions of the column; but typically.

washed ,TEM buffer. a sblutioncontainpH.8; n'Mieth'ylenediamineftetraacetate sulfate is removed:

5) 0.2;,mM. yeast ribonucleic tam dephos horylatedil.

by the actionot'Edcol i alkaline phosphatase v v added with? agitation;,After rninutesfl .rniQ -of v 22:

' and 'centrifu'gedgin a i'elinical centrifuge at T i n s f phos h t dreaction .is' determined phosphate (A151 is released "theenzyrrie'catalytions' of the-. as 'sayy the, amount of: inorganic f phosphatereleasedrisiproportionalto the, enzyme concentration,

for" purpo es ,o-f: ;i llust'r, ation, .the examples illustratingthemethod ofithisz inyenti'onwhich follow are directedito enzymeutiiizedinthe following examples was prepared asfollowsc" 1 2 ass-p e FreshlyharVeStedQdriedQ'M. lysodikticus cells were made. upto a 5% s'uspensionin' 0.01 tris buffer, pH 8.0. The temperature of the suspension-wasbrought to 37 C. and ,crystalline lysozyme was'added to make afinalconcentration of 25 mg. per 100 ml. (an equivalent amountoffresh-or spray-dried egg white may also be used).. The mixture wasstirred slowly for about :10-15 minutes. The lengthof stirring isdependent upon the lysozyme and is determined by the failure of the cellsuspension of a container. v I

A saturated solution of ammonium sulfate (4 C.) was addedto thelysedcells with stirring until the final concentration of ammoniumsulfatewas 33% saturation. The mixture was then centrifuged at 15,000rpm. in a refrigerated centrifuge (4 C.) for minutes. The supernatantliquid'was collected and an additional amount of saturated-ammoniumsulfate solution added until the volume of'TEM buffer, then mentdissolved in a small buffer overnight at 4 C. An

dialy'zed against the same HOlOQis added, the contents" mixed Under thecondito adhere to the walls I '30 bat-h, andv 0.5";ml. of a 0.4%solution of adenosine di- 7. described above in-Example z was: added wita-r ange of-0i2 r0 03 Mupotassium chloride.

I nnat i's llss' wy n r is' on' ar:i1 5,000 rpm, in r rigeratedcentrifuge (4:

butter until the ammonium v I C.), dissolved in TEM buffer arid ,dial yz'ed. against Other methods of preliminary purification may be em-'alciyed,- and itis preferred that quantity ofprotein present; i -the?enzyme preparation 'relativ'e vto the enzyme j activi't'yishoul'gi be;-inaintained as small as possible.

- j'f' rv r eimosrpr NZYMES;

.f theifractionated enzyme prepared as a diethyl- H aminoethylcellulosepolyadehylic"acid column, 0.7 5 by y" washedwith IBM 1 buffer; A 'linear10 cm. previous gradient of: potassium' ehloridej' in ZIEM' buffe-awasapplied in-.0.l; M.- incrementsfiper lliter., Fractions' totaling625ml; were collected and tattle-combined fractions was added 277 g. ofsolid 'until completely dis'solv'ed.'- .Themixture was centrifuged TA LEIf enzyme.preparations-maybe" similarly purified. v I Y, J a "i 1 fontaminating En. R A A QN- N Y as; Numerous;,rriethods havebeen-described in the art m 22 fil A m for thepreparationofpolynucl'eotide phosphoryla'se; The- (a) Pass: kinase -phodiesteraseanimal-Iss0..- 1,350 324" 0.86, 4- DEAE 2,300. 750 1 1.50 DE tE-poiyA 510 v 956 1.30

at 15,000 r.p.m. for '30n'iiriutes-ina refrigerated-centrifuge at 4 C,The sedimentwas.dissolvedain Tbufier and dialyzled for'3 hours againstthe butter to 'yilda final volume of 3.8- m1. polynucl'eotide'phosphorylaseenzyme. The results of this purification are'summariz edin Table -I (a) Turnover number is defined 55 cm moles of adonosinediphosphato converted to poi'yadonyiie acid'in oneminute per 10 g. ofprotein of the I enzyme preparation.

(b) 6280 m 260li1fli'3ti0 of absorption is ameasure of the relativeamount oi-nuoleie'acid and derivatives contaminating the enzymepreparation.

(0) A unitot enzyme is defined as that amount; which will convert 1micromole of ADP to polyadenylic acid in one'minute under standardconditions of optimum substrate concentration.

Example 4, i

Four (4') ml. of the fractionated enzyme preparation prepared asdescribed hereinabove'in Example 2 was applied to a column prepared asin Example 1. The same procedure of purification was carried out. Theresults are summarized in Table 11.

TABLE II Contaminating Turnover Enzymes v e 280 in Number 1 PercentYield I H v Themajor'fraction, of the enzyme appears at" approximately"025 'M.;pot assiu m' 'ainrmimiurit sulfate with stirring Where it isdesired to purify the enzyme preparation without utilizing a preliminaryfractionation as described with reference to the preparation of theenzyme material, the dimensions of the column and the quantityofsubstrate-adsorbent material must be correspondingly increased to takecare of increased contaminants present.

lnsummary this invention provides a method for enzyme purification whichutilizes the specificity of an enzyme for its substrate and theadsorption characteristics of the substrate to separate the desiredenzyme from contaminating enzymes which may also be present.

What is claimed is:

1 A process for purifying an enzyme useful in the synthesis ofpolynucleotides which comprises adsorbing a p'olyribonucleotidesubstrate for said enzyme upon an insoluble material which effectivelybinds said substrate, contacting a solution containing said enzyme withsaid substrate bound to said insoluble material to form anenzyme-substrate complex on said insoluble material and eluting saidenzyme from said enzyme-substrate complex.

2. A process according to claim 1 wherein the enzyme is polynucleotidephosphorylase.

'3. A process according to claim 1 wherein the poly ribonucleotidesubstrate is polyadenylic acid.

4. A process according to claim 1 wherein the insoluble material isdiethylaminoethyl cellulose.

5. A process for purifying polynucleotide phos'phorylase which comprisesadsorbingpolyadenylic acid upon diethylaminoethyl cellulose, contacting'a solution containing polynucleotide phosphorylase with saidpolyadenylic acid bound to said diethylaminoethyl cellulose to form acomplex of said polynucleotide phosphor'ylase with said polyadenylicacid on 'said diethylaminoethyl cellulose and eluting saidpolynucleotide ph'osphorylase from said polynucleotidephosphorylase-polyadenylic acid complex. 7

References Cited by the Examiner A. LOUIS MONACELL, Primary Examiner.

1. THE PROCESS FOR PURIFYING AN ENZYME USEFUL IN THE SYNTHESIS OFPOLYNUCLEOTIDES WHICH COMPRISES ABSORBING A POLYRIBONUCLEOTIDE SUBSTRATEFOR SAID ENZYME UPON AN INSOLUBLE MATERIAL WHICH EFFECTIVELY BINDS SAIDSUBSTRATE, CONTACTING A SOLUTION CONTAINING SAID ENZYME WITH SAIDSUBSTRATE BOUND TO SAID INSOLUBLE MATERIAL TO FORM AN ENZYME-SUBSTRATECOMPLEX ON SAID INSOLUBLE MATERIAL AND ELUTING SAID ENZYME FROM SAIDENZYME-SUBSTRATE COMPLEX.